Na,K-ATPase α2 activity in mammalian skeletal muscle T-tubules is acutely stimulated by extracellular K+

The Na,K-ATPase α2 isoform is the predominant Na,K-ATPase in adult skeletal muscle and the sole Na,K-ATPase in the transverse tubules (T-tubules). In quiescent muscles, the α2 isozyme operates substantially below its maximal transport capacity. Unlike the α1 isoform, the α2 isoform is not required for maintaining resting ion gradients or the resting membrane potential, canonical roles of the Na,K-ATPase in most other cells. However, α2 activity is stimulated immediately upon the start of contraction and, in working muscles, its contribution is crucial to maintaining excitation and resisting fatigue. Here, we show that α2 activity is determined in part by the K⁺ concentration in the T-tubules, through its K⁺ substrate affinity. Apparent K⁺ affinity was determined from measurements of the K_1/2 for K⁺ activation of pump current in intact, voltage-clamped mouse flexor digitorum brevis muscle fibers. Pump current generated by the α2 Na,K-ATPase, Ip, was identified as the outward current activated by K⁺ and inhibited by micromolar ouabain. Ip was outward at all potentials studied (−90 to −30 mV) and increased with depolarization in the subthreshold range, −90 to −50 mV. The Q₁₀ was 2.1 over the range of 22–37°C. The K_1/2,K of Ip was 4.3 ± 0.3 mM at −90 mV and was relatively voltage independent. This K⁺ affinity is lower than that reported for other cell types but closely matches the dynamic range of extracellular K⁺ concentrations in the T-tubules. During muscle contraction, T-tubule luminal K⁺ increases in proportion to the frequency and duration of action potential firing. This K_1/2,K predicts a low fractional occupancy of K⁺ substrate sites at the resting extracellular K⁺ concentration, with occupancy increasing in proportion to the frequency of membrane excitation. The stimulation of preexisting pumps by greater K⁺ site occupancy thus provides a rapid mechanism for increasing α2 activity in working muscles.     

Mechanism of Stimulation and Inhibition of Tonoplast H-ATPase of Beta vulgaris by Chloride and Nitrate

The H⁺-ATPase of tonoplast vesicles isolated from red beet (Beta vulgaris L.) storage tissue was studied with respect to the kinetic effects of Cl⁻ and NO₃⁻. N-Ethylmaleimide (NEM) was employed as a probe to investigate substrate binding and gross conformational changes of the enzyme. Chloride decreased the K_m of the enzyme for ATP but caused relatively little alteration of the V_max. Nitrate increased K_m only. Michaelis-Menten kinetics applied throughout with respect to ATP concentration. Nitrate yielded similar kinetics of inhibition in both the presence and absence of Cl⁻. Other monovalent anions that specifically increased the K_m of the ATPase for ATP were, in order of increasing K_i, SCN⁻, ClO₄⁻, and ClO₃⁻. Sulfate, although inhibitory, manifested noncompetitive kinetics with respect to ATP concentration. ADP, like NO₃⁻, was a competitive inhibitor of the ATPase but ADP and NO₃⁻ did not interact cooperatively nor did either interfere with the inhibitory action of the other. It is concluded that NO₃⁻ does not show competitive kinetics because of its stereochemical similarity to the terminal phosphoryl group of ATP. NEM was an irreversible inhibitor of the tonoplast ATPase. Both Mg·ADP and Mg·ATP protected the enzyme from inactivation by NEM but Mg·ADP was the more potent of the two. Chloride and NO₃⁻ exerted little or no effect on the protective actions of Mg·ADP and Mg·ATP suggesting that neither Cl⁻ nor NO₃⁻ are involved in substrate binding.     

Specific modification of gastric K+-stimulated ATPase activity by thimerosal

Treatment of hog gastric microsomes with the sulfhydryl reagent, thimerosal (ethylmercurithiosalicylate), produced differential effects on the K⁺-ATPase and the K⁺-stimulated p-nitrophenylphosphatase activities. For example, exposure to 2 mM thimerosal for 3 min severely reduced the activity of K⁺-stimulated ATPase, while K⁺-p-nitrophenylphosphatase activity was enhanced 2- to 3-fold. Higher concentration of thimerosal, or longer incubation times, also led to inhibition of K⁺-p-nitrophenylphosphatase. The activated state of p-nitrophenylphosphatase could be sustained by a 20-fold, or greater, dilution of treated membranes, and could be reversed by reduction of membrane SH groups by exogenous thiols. Significant activation of K⁺-p-nitrophenylphosphatase was not produced by p-chloromercuribenzene sulfonate, p-chloromercuribenzoate or mersalyl; however, ethyl mercuric chloride had qualitatively similar activity effects as thimerosal. Kinetics of K⁺-p-nitrophenylphosphatase for thimerosal-treated membranes were altered as follows: V increased; K_m for p-nitrophenylphosphate unchanged for K_a for K⁺ increased. ATP, which is a potent inhibitor of K⁺-p-nitrophenylphosphatase activity in native membranes (K_I ≈ 200 μM). These data suggest that there are multiple SH groups which differentially influence the gastric K⁺-stimulated ATPase activity. Defined treatments with thimerosal are interpreted as an uncoupling of the K⁺-stimulated phosphatase component of the enzyme (for which p-nitrophenylphosphatase is a presumed model reaction). Such differential modifications can be usefully applied to the study of partial reactions of the enzyme and their specific role in the related H⁺-transport reaction.     

Anion interactions with Na,K-ATPase: simultaneous binding of nitrate and eosin

Nucleotide binding affinity to Na,K-ATPase is reduced by a number of anions such as nitrate and perchlorate in comparison with affinity in the presence of chloride (all with sodium as the cation). The reduction correlates with the position of these anions in the Hofmeister series. Transient kinetic experiments using the fluorescent dye eosin—which binds to the nucleotide site of the Na,K-ATPase—show that simultaneous anion binding, exemplified with nitrate, and eosin binding is possible. The effect of nitrate on eosin binding is reflected in a decreased binding-rate constant and an increased dissociation rate constant, leading to a decreased equilibrium binding constant for eosin. Since eosin binding is analogous with nucleotide binding to Na,K-ATPase, the results suggest the simultaneous presence of nucleotide and anion binding sites.Abbreviations E₁ the protein conformation in Na⁺ - E₂ the enzyme conformation in K⁺ - Eo eosin (tetrabromofluorescein) - F fluorescence - I ionic strength - k_i rate constant - K_i equilibrium dissociation constant - K_i,0 equilibrium dissociation constant at zero ionic strength - N nitrate - z_i net charge - charge product z_i·z_j     

A novel thermostable arylesterase from the archaeon Sulfolobus solfataricus P1: purification, characterization, and expression

A novel thermostable arylesterase, a 35-kDa monomeric enzyme, was purified from the thermoacidophilic archaeon Sulfolobus solfataricus P1. The optimum temperature and pH were 94°C and 7.0, respectively. The enzyme displayed remarkable thermostability: it retained 52% of its activity after 50 h of incubation at 90°C. In addition, the purified enzyme showed high stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme has broad substrate specificity besides showing an arylesterase activity toward aromatic esters: it exhibits not only carboxylesterase activity toward tributyrin and p-nitrophenyl esters containing unsubstituted fatty acids from butyrate (C₄) to palmitate (C₁₆), but also paraoxonase activity toward organophosphates such as p-nitrophenylphosphate, paraoxon, and methylparaoxon. The k_cat/K_m ratios of the enzyme for phenyl acetate and paraoxon, the two most preferable substrates among all tested, were 30.6 and 119.4 s⁻¹·μM⁻¹, respectively. The arylesterase gene consists of 918 bp corresponding to 306 amino acid residues. The deduced amino acid sequence shares 34% identity with that of arylesterase from Acinetobacter sp. strain ADP1. Furthermore, we successfully expressed active recombinant S. solfataricus arylesterase in Escherichia coli. Together, our results show that the enzyme is a serine esterase belonging to the A-esterases and contains a catalytic triad composed of Ser156, Asp251, and His281 in the active site.     

Osmotic stress and viscous retardation of the Na,K-ATPase ion pump

The transport function of the Na pump (Na,K-ATPase) in cellular ion homeostasis involves both nucleotide binding reactions in the cytoplasm and alternating aqueous exposure of inward- and outward-facing ion binding sites. An osmotically active, nonpenetrating polymer (poly(ethyleneglycol); PEG) and a modifier of the aqueous viscosity (glycerol) were used to probe the overall and partial enzymatic reactions of membranous Na,K-ATPase from shark salt glands. Both inhibit the steady-state Na,K-ATPase as well as Na-ATPase activity, whereas the K⁺-dependent phosphatase activity is little affected by up to 50% of either. Both Na,K-ATPase and Na-ATPase activities are inversely proportional to the viscosity of glycerol solutions in which the membranes are suspended, in accordance with Kramers’ theory for strong coupling of fluctuations at the active site to solvent mobility in the aqueous environment. PEG decreases the affinity for Tl⁺ (a congener for K⁺), whereas glycerol increases that for the nucleotides ATP and ADP in the presence of NaCl but has little effect on the affinity for Tl⁺. From the dependence on osmotic stress induced by PEG, the aqueous activation volume for the Na,K-ATPase reaction is estimated to be ∼5-6 nm³ (i.e., ∼180 water molecules), approximately half this for Na-ATPase, and essentially zero for p-nitrophenol phosphatase. The change in aqueous hydrated volume associated with the binding of Tl⁺ is in the region of 9 nm³. Analysis of 15 crystal structures of the homologous Ca-ATPase reveals an increase in PEG-inaccessible water space of ∼22 nm³ between the E₁-nucleotide bound forms and the E₂-thapsigargin forms, showing that the experimental activation volumes for Na,K-ATPase are of a magnitude comparable to the overall change in hydration between the major E₁ and E₂ conformations of the Ca-ATPase.     

Probing tRNA interaction with biogenic polyamines

Biogenic polyamines are found to modulate protein synthesis at different levels. This effect may be explained by the ability of polyamines to bind and influence the secondary structure of tRNA, mRNA, and rRNA. We report the interaction between tRNA and the three biogenic polyamines putrescine, spermidine, spermine, and cobalt(III)hexamine at physiological conditions, using FTIR spectroscopy, capillary electrophoresis, and molecular modeling. The results indicated that tRNA was stabilized at low biogenic polyamine concentration, as a consequence of polyamine interaction with the backbone phosphate group. The main tRNA reactive sites for biogenic polyamine at low concentration were guanine-N7/O6, uracil-O2/O4, adenine-N3, and 2′OH of the ribose. At high polyamine concentration, the interaction involves guanine-N7/O6, adenine-N7, uracil-O2 reactive sites, and the backbone phosphate group. The participation of the polycation primary amino group, in the interaction and the presence of the hydrophobic contact, are also shown. The binding affinity of biogenic polyamine to tRNA molecule was in the order of spermine > spermidine > putrescine with K_Spm = 8.7 × 10⁵ M⁻¹, K_Spd = 6.1 × 10⁵ M⁻¹, and K_Put = 1.0 × 10⁵ M⁻¹, which correlates with their positively charged amino group content. Hill analysis showed positive cooperativity for the biogenic polyamines and negative cooperativity for cobalt-hexamine. Cobalt(III)hexamine contains high- and low-affinity sites in tRNA with K₁ = 3.2 × 10⁵ M⁻¹ and K₂ = 1.7 × 10⁵ M⁻¹, that have been attributed to the interactions with guanine-N7 sites and the backbone PO₂ group, respectively. This mechanism of tRNA binding could explain the condensation phenomenon observed at high Co(III) content, as previously shown in the Co(III)–DNA complexes.     

Transporting Antitumor Drug Tamoxifen and Its Metabolites, 4-Hydroxytamoxifen and Endoxifen by Chitosan Nanoparticles

Synthetic and natural polymers are often used as drug delivery systems in vitro and in vivo. Biodegradable chitosan of different sizes were used to encapsulate antitumor drug tamoxifen (Tam) and its metabolites 4-hydroxytamoxifen (4-Hydroxytam) and endoxifen (Endox). The interactions of tamoxifen and its metabolites with chitosan 15, 100 and 200 KD were investigated in aqueous solution, using FTIR, fluorescence spectroscopic methods and molecular modeling. The structural analysis showed that tamoxifen and its metabolites bind chitosan via both hydrophilic and hydrophobic contacts with overall binding constants of K _tam-ch-15  = 8.7 (±0.5)×10³ M⁻¹, K _tam-ch-100  = 5.9 (±0.4)×10⁵ M⁻¹, K _tam-ch-200  = 2.4 (±0.4)×10⁵ M⁻¹ and K _{hydroxytam-ch-15}  = 2.6(±0.3)×10⁴ M⁻¹, K _{hydroxytam – ch-100}  = 5.2 (±0.7)×10⁶ M⁻¹ and K _{hydroxytam-ch-200}  = 5.1 (±0.5)×10⁵ M⁻¹, K _endox-ch-15  = 4.1 (±0.4)×10³ M⁻¹, K _endox-ch-100  = 1.2 (±0.3)×10⁶ M⁻¹ and K _endox-ch-200  = 4.7 (±0.5)×10⁵ M⁻¹ with the number of drug molecules bound per chitosan (n) 2.8 to 0.5. The order of binding is ch-100>200>15 KD with stronger complexes formed with 4-hydroxytamoxifen than tamoxifen and endoxifen. The molecular modeling showed the participation of polymer charged NH₂ residues with drug OH and NH₂ groups in the drug-polymer adducts. The free binding energies of −3.46 kcal/mol for tamoxifen, −3.54 kcal/mol for 4-hydroxytamoxifen and −3.47 kcal/mol for endoxifen were estimated for these drug-polymer complexes. The results show chitosan 100 KD is stronger carrier for drug delivery than chitosan-15 and chitosan-200 KD.     

Electrophysiological Characterization of the Rat Epithelial Na+ Channel (rENaC) Expressed in MDCK Cells : Effects of Na+ and Ca2+

The epithelial Na⁺ channel (ENaC), composed of three subunits (α, β, and γ), is expressed in several epithelia and plays a critical role in salt and water balance and in the regulation of blood pressure. Little is known, however, about the electrophysiological properties of this cloned channel when expressed in epithelial cells. Using whole-cell and single channel current recording techniques, we have now characterized the rat αβγENaC (rENaC) stably transfected and expressed in Madin-Darby canine kidney (MDCK) cells. Under whole-cell patch-clamp configuration, the αβγrENaC-expressing MDCK cells exhibited greater whole cell Na⁺ current at −143 mV (−1,466.2 ± 297.5 pA) than did untransfected cells (−47.6 ± 10.7 pA). This conductance was completely and reversibly inhibited by 10 μM amiloride, with a Ki of 20 nM at a membrane potential of −103 mV; the amiloride inhibition was slightly voltage dependent. Amiloride-sensitive whole-cell current of MDCK cells expressing αβ or αγ subunits alone was −115.2 ± 41.4 pA and −52.1 ± 24.5 pA at −143 mV, respectively, similar to the whole-cell Na⁺ current of untransfected cells. Relaxation analysis of the amiloride-sensitive current after voltage steps suggested that the channels were activated by membrane hyperpolarization. Ion selectivity sequence of the Na⁺ conductance was Li⁺ > Na⁺ >> K⁺ = N-methyl-d-glucamine⁺ (NMDG⁺). Using excised outside-out patches, amiloride-sensitive single channel conductance, likely responsible for the macroscopic Na⁺ channel current, was found to be ∼5 and 8 pS when Na⁺ and Li⁺ were used as a charge carrier, respectively. K⁺ conductance through the channel was undetectable. The channel activity, defined as a product of the number of active channel (n) and open probability (P _o), was increased by membrane hyperpolarization. Both whole-cell Na⁺ current and conductance were saturated with increased extracellular Na⁺ concentrations, which likely resulted from saturation of the single channel conductance. The channel activity (nP _o) was significantly decreased when cytosolic Na⁺ concentration was increased from 0 to 50 mM in inside-out patches. Whole-cell Na⁺ conductance (with Li⁺ as a charge carrier) was inhibited by the addition of ionomycin (1 μM) and Ca²⁺ (1 mM) to the bath. Dialysis of the cells with a pipette solution containing 1 μM Ca²⁺ caused a biphasic inhibition, with time constants of 1.7 ± 0.3 min (n = 3) and 128.4 ± 33.4 min (n = 3). An increase in cytosolic Ca²⁺ concentration from <1 nM to 1 μM was accompanied by a decrease in channel activity. Increasing cytosolic Ca²⁺ to 10 μM exhibited a pronounced inhibitory effect. Single channel conductance, however, was unchanged by increasing free Ca²⁺ concentrations from <1 nM to 10 μM. Collectively, these results provide the first characterization of rENaC heterologously expressed in a mammalian epithelial cell line, and provide evidence for channel regulation by cytosolic Na⁺ and Ca²⁺.     

Characterization of K+-dependent and K+-independentp-nitrophenylphosphatase activity of synaptosomes

These experiments examined effects of several ligands on the K⁺ p-nitrophenylphosphatase activity of the (Na⁺,K⁺)-ATPase in membranes of a rat brain cortex synaptosomal preparation. K⁺-independent hydrolysis of this substrate by the synaptosomal preparation was studied in parallel; the rate of hydrolysis in the absence of K⁺ was approximately 75% less than that observed when K⁺ was included in the incubation medium. The response to the H⁺ concentrations was different: K⁺-independent activity showed a pH optimum around 6.5–7.0, while the K⁺-dependent activity was relatively low at this pH range. Ouabain (0.1 mM) inhibited K⁺-dependent activity 50%; a concentration 10 times higher did not produce any appreciable effect on the K⁺-independent activity. Na⁺ did not affect K⁺-independent activity at all, while the same ligand concentration inhibited sharply the K⁺-dependent activity; this inhibition was not competitive with the substrate,p-nitrophenyl phosphate. K⁺-dependent activity was stimulated by Mg²⁺ with low affinity (millimolar range), and 3 mM Mg²⁺ produced a slight stimulation of the activity in absence of K⁺, which could be interpreted as Mg²⁺ occupying the K⁺ sites. Ca²⁺ had no appreciable effect on the activity in the absence of K⁺. However, in the presence of K⁺ a sharp inhibition was found with all Ca²⁺ concentrations studied. ATP (0.5 mM) did not affect the K⁺-independent activity, but this nucleotide behaved as a competitive inhibitor top-nitrophenylphosphate. Pi inhibited activity in the presence of K⁺, competively to the substrate, so it could be considered as the second product of the reaction sequence.Abbreviations used p-NPP p-nitrophenylphosphate - p-NPPase rho-nitrophenylphosphatase activity     

Phanerochaete chrysosporium β-Glucosidases: Induction, Cellular Localization, and Physical Characterization

Phanerochaete chrysosporium produces intracellular soluble and particulate β-glucosidases and an extracellular β-glucosidase. The extracellular enzyme is induced by cellulose but repressed in the presence of glucose. The molecular weight of this enzyme is 90,000. The K_m for p-nitrophenyl-β-glucoside is 1.6 × 10⁻⁴ M; the K_i for glucose, a competitive inhibitor, is 5.0 × 10⁻⁴ M. The K_m for cellobiose is 5.3 × 10⁻⁴ M. The intracellular soluble enzyme is induced by cellobiose; this induction is prevented by cycloheximide. The presence of 300 mM glucose in the medium, however, had no effect on induction. The K_m for p-nitrophenyl-β-glucoside is 1.1 × 10⁻⁴ M. The molecular weight of this enzyme is ~410,000. Both enzymes have an optimal temperature of 45°C and an E_act of 9.15 kcal (ca. 3.83 × 10⁴ J). The pH optima, however, were ~7.0 and 5.5 for the intracellular and extracellular enzymes, respectively.     

NADPH–Cytochrome P450 Reductase Mediates the Fatty Acid Desaturation of ω3 and ω6 Desaturases from Mortierella alpina

Fatty acid desaturases play an important role in maintaining the appropriate structure and function of biological membranes. The biochemical characterization of integral membrane desaturases, particularly ω3 and ω6 desaturases, has been limited by technical difficulties relating to the acquisition of large quantities of purified proteins, and by the fact that functional activities of these proteins were only tested in an NADH-initiated reaction system. The main aim of this study was to reconstitute an NADPH-dependent reaction system in vitro and investigate the kinetic properties of Mortierella alpina ω3 and ω6 desaturases in this system. After expression and purification of the soluble catalytic domain of NADPH–cytochrome P450 reductase, the NADPH-dependent fatty acid desaturation was reconstituted for the first time in a system containing NADPH, NADPH–cytochrome P450 reductase, cytochrome b5, M. alpina ω3 and ω6 desaturase and detergent. In this system, the maximum activity of ω3 and ω6 desaturase was 213.4 ± 9.0 nmol min⁻¹ mg⁻¹ and 10.0 ± 0.5 nmol min⁻¹ mg⁻¹, respectively. The highest k_cat/K_m value of ω3 and ω6 desaturase was 0.41 µM⁻¹ min⁻¹ and 0.09 µM⁻¹ min⁻¹ when using linoleoyl CoA (18:2 ω6) and oleoyl CoA (18:1 ω9) as substrates, respectively. M. alpina ω3 and ω6 desaturases were capable of using NADPH as reductant when mediated by NADPH–cytochrome P450 reductase; although, their efficiency is distinguishable from NADH-dependent desaturation. These results provide insights into the mechanisms underlying ω3 and ω6 fatty acid desaturation and may facilitate the production of important fatty acids in M. alpina.     

The Epac1 Signaling Pathway Regulates Cl? Secretion via Modulation of Apical KCNN4c Channels in Diarrhea

The apical membrane of intestinal epithelia expresses intermediate conductance K⁺ channel (KCNN4), which provides the driving force for Cl⁻ secretion. However, its role in diarrhea and regulation by Epac1 is unknown. Previously we have established that Epac1 upon binding of cAMP activates a PKA-independent mechanism of Cl⁻ secretion via stimulation of Rap2-phospholipase Cϵ-[Ca²⁺]_i signaling. Here we report that Epac1 regulates surface expression of KCNN4c channel through its downstream Rap1A-RhoA-Rho-associated kinase (ROCK) signaling pathway for sustained Cl⁻ secretion. Depletion of Epac1 protein and apical addition of TRAM-34, a specific KCNN4 inhibitor, significantly abolished cAMP-stimulated Cl⁻ secretion and apical K⁺ conductance (I_K(ap)) in T84WT cells. The current-voltage relationship of basolaterally permeabilized monolayers treated with Epac1 agonist 8-(4-chlorophenylthio)-2′-O- methyladenosine 3′,5′-cyclic monophosphate showed the presence of an inwardly rectifying and TRAM-34-sensitive K⁺ channel in T84WT cells that was absent in Epac1KDT84 cells. Reconstructed confocal images in Epac1KDT84 cells revealed redistribution of KCNN4c proteins into subapical intracellular compartment, and a biotinylation assay showed ∼83% lower surface expression of KCNN4c proteins compared with T84WT cells. Further investigation revealed that an Epac1 agonist activates Rap1 to facilitate I_K(ap). Both RhoA inhibitor (GGTI298) and ROCK inhibitor (H1152) significantly reduced cAMP agonist-stimulated I_K(ap), whereas the latter additionally reduced colocalization of KCNN4c with the apical membrane marker wheat germ agglutinin in T84WT cells. In vivo mouse ileal loop experiments showed reduced fluid accumulation by TRAM-34, GGTI298, or H1152 when injected together with cholera toxin into the loop. We conclude that Rap1A-dependent signaling of Epac1 involving RhoA-ROCK is an important regulator of intestinal fluid transport via modulation of apical KCNN4c channels, a finding with potential therapeutic value in diarrheal diseases.     

Potassium Currents in Freshly Dissociated Uterine Myocytes from Nonpregnant and Late-Pregnant Rats

In freshly dissociated uterine myocytes, the outward current is carried by K⁺ through channels highly selective for K⁺. Typically, nonpregnant myocytes have rather noisy K⁺ currents; half of them also have a fast-inactivating transient outward current (I_TO). In contrast, the current records are not noisy in late pregnant myocytes, and I_TO densities are low. The whole-cell I_K of nonpregnant myocytes respond strongly to changes in [Ca²⁺]_o or changes in [Ca²⁺]_i caused by photolysis of caged Ca²⁺ compounds, nitr 5 or DM-nitrophene, but that of late-pregnant myocytes respond weakly or not at all. The Ca²⁺ insensitivity of the latter is present before any exposure to dissociating enzymes. By holding at −80, −40, or 0 mV and digital subtractions, the whole-cell I_K of each type of myocyte can be separated into one noninactivating and two inactivating components with half-inactivation at approximately −61 and −22 mV. The noninactivating components, which consist mainly of iberiotoxin-susceptible large-conductance Ca²⁺-activated K⁺ currents, are half-activated at 39 mV in nonpregnant myocytes, but at 63 mV in late-pregnant myocytes. In detached membrane patches from the latter, identified 139 pS, Ca²⁺-sensitive K⁺ channels also have a half-open probability at 68 mV, and are less sensitive to Ca²⁺ than similar channels in taenia coli myocytes. Ca²⁺-activated K⁺ currents, susceptible to tetraethylammonium, charybdotoxin, and iberiotoxin contribute 30–35% of the total I_K in nonpregnant myocytes, but <20% in late-pregnant myocytes. Dendrotoxin-susceptible, small-conductance delayed rectifier currents are not seen in nonpregnant myocytes, but contribute ∼20% of total I_K in late-pregnant myocytes. Thus, in late-pregnancy, myometrial excitability is increased by changes in K⁺ currents that include a suppression of the I_TO, a redistribution of I_K expression from large-conductance Ca²⁺-activated channels to smaller-conductance delayed rectifier channels, a lowered Ca²⁺ sensitivity, and a positive shift of the activation of some large-conductance Ca²⁺-activated channels.     

Characterization of the Catalytic and Nucleotide Binding Properties of the α-Kinase Domain of Dictyostelium Myosin-II Heavy Chain Kinase A

The α-kinases are a widely expressed family of serine/threonine protein kinases that exhibit no sequence identity with conventional eukaryotic protein kinases. In this report, we provide new information on the catalytic properties of the α-kinase domain of Dictyostelium myosin-II heavy chain kinase-A (termed A-CAT). Crystallization of A-CAT in the presence of MgATP yielded structures with AMP or adenosine in the catalytic cleft together with a phosphorylated Asp-766 residue. The results show that the β- and α-phosphoryl groups are transferred either directly or indirectly to the catalytically essential Asp-766. Biochemical assays confirmed that A-CAT hydrolyzed ATP, ADP, and AMP with k_cat values of 1.9, 0.6, and 0.32 min⁻¹, respectively, and showed that A-CAT can use ADP to phosphorylate peptides and proteins. Binding assays using fluorescent 2′/3′-O-(N-methylanthraniloyl) analogs of ATP and ADP yielded K_d values for ATP, ADP, AMP, and adenosine of 20 ± 3, 60 ± 20, 160 ± 60, and 45 ± 15 μm, respectively. Site-directed mutagenesis showed that Glu-713, Leu-716, and Lys-645, all of which interact with the adenine base, were critical for nucleotide binding. Mutation of the highly conserved Gln-758, which chelates a nucleotide-associated Mg²⁺ ion, eliminated catalytic activity, whereas loss of the highly conserved Lys-722 and Arg-592 decreased k_cat values for kinase and ATPase activities by 3–6-fold. Mutation of Asp-663 impaired kinase activity to a much greater extent than ATPase, indicating a specific role in peptide substrate binding, whereas mutation of Gln-768 doubled ATPase activity, suggesting that it may act to exclude water from the active site.     

Sequential potassium binding at the extracellular side of the Na,K-Pump

Ion binding at the extracellular face of the Na,K-ATPase is electrogenic and can be monitored by the styryl dye RH 421 in membrane fragments containing a high density of the Na,K-pumps. The fluorescent probe is noncovalently bound to the membrane and responds to changes of the local electric field generated by binding or release of cations inside the protein. Due to the fact that K⁺ binding from the extracellular side is an electrogenic reaction, it is possible to detect the amount of ions bound to the pump as function of the aqueous concentration. The results are in contradiction to a second order reaction, i.e., a simultaneous binding of two K⁺ ions. A mathematical model is presented to discuss the nature of the two step binding process. On the basis of this model the data allow a quantitative distinction between binding of the first and the second K⁺ ion. The temperature dependence of ion binding has been investigated. At low temperatures the apparent dissociation constants differ significantly. In the temperature range above 20°C the resulting apparent dissociation constants for both K⁺ ions merge and have values between 0.2 and 0.3 mm, which is consistent with previous experiments. The activation energy for the half saturating concentration of K⁺ is 22 kJ/mol. Additional analysis of the titration curve of K⁺ binding to the state P — E₂ by the Hill equation yields a Hill coefficient, n_Hill, of 1.33, which is in agreement with previously published data.The authors would like to thank G. Witz for technical assistance. This work has been financially supported by the Deutsche Forschungsgemeinschaft (SFB 156).     

Imperatoxin A Induces Subconductance States in Ca2+ Release Channels (Ryanodine Receptors) of Cardiac and Skeletal Muscle

Single-channel and [³H]ryanodine binding experiments were carried out to examine the effects of imperatoxin activator (IpTx_a), a 33 amino acid peptide isolated from the venom of the African scorpion Pandinus imperator, on rabbit skeletal and canine cardiac muscle Ca²⁺ release channels (CRCs). Single channel currents from purified CRCs incorporated into planar lipid bilayers were recorded in 250 mM KCl media. Addition of IpTx_a in nanomolar concentration to the cytosolic (cis) side, but not to the lumenal (trans) side, induced substates in both ryanodine receptor isoforms. The substates displayed a slightly rectifying current–voltage relationship. The chord conductance at −40 mV was ∼43% of the full conductance, whereas it was ∼28% at a holding potential of +40 mV. The substate formation by IpTx_a was voltage and concentration dependent. Analysis of voltage and concentration dependence and kinetics of substate formation suggested that IpTx_a reversibly binds to the CRC at a single site in the voltage drop across the channel. The rate constant for IpTx_a binding to the skeletal muscle CRC increased e-fold per +53 mV and the rate constant of dissociation decreased e-fold per +25 mV applied holding potential. The effective valence of the reaction leading to the substate was ∼1.5. The IpTx_a binding site was calculated to be located at ∼23% of the voltage drop from the cytosolic side. IpTx_a induced substates in the ryanodine-modified skeletal CRC and increased or reduced [³H]ryanodine binding to sarcoplasmic reticulum vesicles depending on the level of channel activation. These results suggest that IpTx_a induces subconductance states in skeletal and cardiac muscle Ca²⁺ release channels by binding to a single, cytosolically accessible site different from the ryanodine binding site.     

Kinesin-2 KIF3AB Exhibits Novel ATPase Characteristics

KIF3AB is an N-terminal processive kinesin-2 family member best known for its role in intraflagellar transport. There has been significant interest in KIF3AB in defining the key principles that underlie the processivity of KIF3AB in comparison with homodimeric processive kinesins. To define the ATPase mechanism and coordination of KIF3A and KIF3B stepping, a presteady-state kinetic analysis was pursued. For these studies, a truncated murine KIF3AB was generated. The results presented show that microtubule association was fast at 5.7 μm⁻¹ s⁻¹, followed by rate-limiting ADP release at 12.8 s⁻¹. ATP binding at 7.5 μm⁻¹ s⁻¹ was followed by an ATP-promoted isomerization at 84 s⁻¹ to form the intermediate poised for ATP hydrolysis, which then occurred at 33 s⁻¹. ATP hydrolysis was required for dissociation of the microtubule·KIF3AB complex, which was observed at 22 s⁻¹. The dissociation step showed an apparent affinity for ATP that was very weak (K_½,ATP at 133 μm). Moreover, the linear fit of the initial ATP concentration dependence of the dissociation kinetics revealed an apparent second-order rate constant at 0.09 μm⁻¹ s⁻¹, which is inconsistent with fast ATP binding at 7.5 μm⁻¹ s⁻¹ and a K_d,ATP at 6.1 μm. These results suggest that ATP binding per se cannot account for the apparent weak K_½,ATP at 133 μm. The steady-state ATPase K_m,ATP, as well as the dissociation kinetics, reveal an unusual property of KIF3AB that is not yet well understood and also suggests that the mechanochemistry of KIF3AB is tuned somewhat differently from homodimeric processive kinesins.     

Polymorphism in miR-31 and miR-584 binding site in the angiotensinogen gene differentially influences body fat distribution in both sexes

Angiotensinogen (AGT), its active fragments and microRNA-31 (miR-31) play an important role in adipocyte differentiation. AGT contains a miR-31 polymorphic binding site. We hypothesize that the rs7079 polymorphism in the miR-31/584 binding site of the AGT gene could influence body fat distribution. A total of 751 subjects (195 men, 556 women) were enrolled in the study. The rs7079 genotypes were determined by qRT-PCR. Anthropometric measurements were taken on all subjects, who were subsequently divided into two groups: obese (>30 kg m⁻²) and non-obese (<30 kg m⁻²). Linear regression models were created to determine the contributions of sex, obesity status and rs7079 to all measured parameters. Adding the rs7079 genotype significantly contributed to the linear regression model for waist circumference (p = 0.013), hip circumference (p = 0.018) and supraspinal skin-fold thickness (p = 1 × 10⁻³). Differences between sexes and between the obese and non-obese groups were observed. Waist circumference was lower in men carrying the A allele (p = 0.022); hip circumference was higher only in obese women carrying the A allele (p = 0.015). While men carrying the A allele had lower supraspinal skin-fold thickness (p = 0.022), this parameter was found to be higher in A allele carrying women (p = 3 × 10⁻³). The higher total sum of skin-fold thickness in A allele carrying women was restricted to obese individuals (p = 0.028). The presence of the A allele was associated with both lower tricipital skin-fold thickness in non-obese women (p = 0.023) and a trend of higher thickness in non-obese men (p = 0.065). Significant associations of rs7079 in the AGT gene and body fat distribution were observed. The distribution followed opposing patterns in both sexes.